T cells that are specific for individual antigens (peptides) of HIV usually occur at low frequencies in vivo and unambiguous characterizing them is an ongoing concern. The full range of HIV peptides (determinants) targeted by such memory cells is not known, nor is the magnitude and quality of the T cell response to these determinants fully understood. We propose to use here a new generation of image-analysis-assisted ELISPOT assays to characterize at single-cell resolution the HIV antigen-specific CD4 and CD8 cell activity in HIV-infected patients, their contact persons, and healthy donors that have not been exposed to HIV. The first hypothesis to be tested is that using a complete set of overlapping 10-mer Gag peptides that walk the sequence in steps of single amino acids (501 peptides) in conjunction with two-color IFN-gamma/IL-4 ELISPOT analysis is a suitable method for measuring the frequencies of all memory T cells specific for Gag in any individual (irrespective of HLA-type) and detecting the full range of peptide reactivity in that individual (Specific Aim 1). Next, we wish to test the hypothesis that such ELISPOT assays, including the one for peptide-induced direct ex vivo granzyme B, are suited to measure and characterize Gag-specific CD8 and CD4 cells (Aim 2). By performing two-color ELISPOT assays at the single-cell level to assess the co-expression of cytokine pairs IL-2, IL-4, IL-5, IL-10, IFN-gamma, and TNF, we wish to test the third hypothesis, that cytokine expressed by human T cells is much more highly regulated than predicted by the Th1/Th2, Tc1/Tc2 model (Aim 3). By defining repeatedly, over the 5 years, in individual HIV-infected patients, the set of Gag peptides their T cells actively target and the number of T cells targeting each peptide, we wish to test the hypothesis that the anti-HIV-reactive T cell repertoire is dynamic (Aim 4).